The ultimate goal of this research project is to develop a detailed understanding of how the coenzyme form of vitamin B12 is activated via carbon-cobalt bond homolysis by coenzyme B12-requiring enzymes. Recent work suggests that homolysis of thermally labile alkylcobalamins (benzyl- and neopentylcobalamin) is driven by steric interactions between the organic ligand and the upward projecting a, c, and g acetamide side chains. Should this hypothesis prove correct, a mechanism for enzymatic activation of 5' -deoxy- adenosylcobalamin in which the enzyme increases the steric interactions of the 5'-deoxyadenosyl ligand with the acetamide side chains would become compelling. The proposed research is designated to rigorously test the hypothesis that carbon cobalt bond homolysis in such alkylcobalamins is the result of steric interactions between the acetamides and the organic ligand. This hypothesis will be tested by two types of studies. First, the thermal homolysis of alkylcobalamin analogs in which the steric bulk and/or the number of upward projecting side chains has been altered chemically will be studied. Second, the importance of acetamide side chain mobility on carbon-cobalt bond homolysis will be studied by capitalizing on our recent observation that a B12 binding protein (haptoccorrin) from chicken serum stabilizes the carbon-cobalt bond of thermaly labile alkylcobalamins by some three orders of magnitude. We have postulated that the binding of cobalamins to haptocorrin involves hydrogen bonding of the side chain amides to acceptors in the protein binding pocket and that the consequent thermal immobilization of the acetamides is responsible for the observed stabilization. Hence, the thermolysis of alkylcobalamin analogs in which the hydrogen bonding ability of the upward directed side chains to the protein has been reduced should show reduced stabilization of the Co-C bond upon binding to haptocorrin. Such derivatives will include the a, c, and g N- methylamides and monocarboxylates, the C8 and C13 epimers (in which the d or e side chain projects upward) as well as other analogs which may yield altered steric interactions with the organic ligand. The importance of hydrogen bonding in the association of cobalamins with haptocorrin will be determined by studies of the thermodynamics of the binding reactions of cabalamins and their analogs from which specific hydrogen bond donors have been removed chemically. Additional evidence will be sought from 1H and 15N NMR studies of the complex of haptocorrin with cyanocobalamin uniformly enriched in 15N obtained by fermentation using Streptomyces griseus. These measurements will include polarization transfer and multiple quantum coherence spectroscopy as well as 15N- spectral edited 1H NMR. These experiments will permit observation of the chemical shifts of the amide nitrogens and protons as well as the proton chemical shift thermal gradients and H-D exchange rates.